An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.

Macrolide-resistant Mycoplasma pneumoniae in adults in Zhejiang, China.

Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistant M. pneumoniae has been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistant M. pneumoniae infection in adults. In this study, we survey the macrolide-resistant M. pneumoniae in adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated for M. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistant M. pneumoniae isolates were also investigated in vitro. Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) of M. pneumoniae strains isolated from adults with CAP were resistant to erythromycin (MIC=128 to >256 µg/ml), clarithromycin (MIC=128 to >256 µg/ml), and azithromycin (MIC=32 to >64 µg/ml). Furthermore, all macrolide-resistant M. pneumoniae strains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistant M. pneumoniae infection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistant M. pneumoniae in the future.

Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas.

During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions.

Discovery of the Clinical Candidate MAK683: An EED-Directed, Allosteric, and Selective PRC2 Inhibitor for the Treatment of Advanced Malignancies.

Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.

Plasma cellular prion protein concentrations correlate with severity and prognosis of aneurysmal subarachnoid hemorrhage.

BACKGROUND: Cellular prion protein (PrPc) is greatly expressed in injured brain tissues. We investigates correlation of plasma PrPc concentrations with severity, delayed cerebral ischemia (DCI) plus prognosis following aneurysmal subarachnoid hemorrhage (aSAH). METHODS: Plasma PrPc concentrations were measured in 110 aSAH patients and 110 healthy controls. The World Federation of Neurological Surgeons scale (WFNS) score, Glasgow coma scale (GCS) score, Hunt-Hess score and modified Fisher score were utilized to assess hemorrhagic severity. Relations of plasma PrPc concentrations to DCI and 90-day poor outcome (Glasgow outcome scale score of 1-3) were analyzed using multivariate analysis. Prognostic predictive capabilities were determined under receiver operating characteristic curve. RESULTS: Plasma PrPc concentrations were significantly higher in patients than in controls. Plasma PrPc concentrations were tightly correlated with WFNS score, GCS score, Hunt-Hess score and modified Fisher score. Plasma PrPc emerged as an independent predictor for 90-day poor outcome, but not for DCI. Plasma PrPc concentrations exhibited similar prognostic predictive abilities, as compared to WFNS score, GCS score, Hunt-Hess score and modified Fisher score. CONCLUSIONS: Plasma PrPc concentrations are highly associated with severity and poor outcome after hemorrhagic stroke, indicating that plasma PrPc may serve as a useful prognostic biomarker for aSAH.

[Real-time fluorescence quantitative polymerase chain reaction detection for Toxoplasma gondii B1 gene in mice urine].

A pair of specific primers and a TaqMan probe were designed based on the sequence of Toxoplasma gondii B1 gene from GenBank database. Total DNA of T. gondii was extracted from fresh mice urine. DNA fragment of B1 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. Following identification, the positive recombinant plasmid was used as reference template to generate standard curve and melt curve. Sensitivity, reproducibility, linear range and stability of reference plasmids were determined. The sensitivity of this method was 10(4) copies/ml. The coefficient of variation (cv) of intra-assay and inter-assay were 2.42% and 4.18%, respectively. Linear range was (10(3)-10(7)) copies/ml. The specificity was 100%. The reference materials were stable. Real-time FQ-PCR of T. gondii DNA in mice urine has been constructed, which is a convenient, sensitive and reliable method for quantifying T. gondii DNA in mice urine.

[Research progress on Th17 cells].

Th17(T helper 17 cell), a newly discovered subset of T cells is associated with IL-23 and characterized by production of IL-17, the functions of which are distinct from those of Th1, Th2 and Treg subsets. The development of Th17 cells can be promoted by TGF-beta1, IL-6, and IL-23; but inhibited by IFN-gamma, IL-4 and Socs3. It is clear that Th17 cells have protective effects on body by facilitating the pro-inflammatory responses. On the other hand, the role of Th17 cells in the pathophysiology of autoimmune diseases has been described.

Induction of Protective Immunity against Toxoplasma gondii in Mice by Nucleoside Triphosphate Hydrolase-II (NTPase-II) Self-amplifying RNA Vaccine Encapsulated in Lipid Nanoparticle (LNP).

RNA-based vaccine represents an irresistible and safe immunization strategy with decreasing theoretical risks of genomic integration and malignant cell transformation. To our knowledge, however, there is no report about development of RNA vaccine against Toxoplasma gondii infection. We have previously demonstrated that the recombinant T. gondii nucleoside triphosphate hydrolase-II (NTPase-II) protein is able to provide protective Th1 cell-mediated immunity against T. gondii. Herein, we evaluated the immunogenic potential of a self-amplifying RNA vaccine-encoding T. gondii NTPase-II gene, RREP-NTPase-II, delivered by a synthetic lipid nanoparticle (LNP). Immunization of mice with naked RREP-NTPase-II induced a strong cellular and humoral immune response with high-IgG antibody titers and IFN-γ production. The immunized mice displayed significantly prolonged survival time and reduction in brain parasite load (46.4%) compared with control group. Furthermore, mice vaccinated with RREP-NTPase-II-encapsulated LNP displayed significantly enhanced protection against acute infection as well as chronic infection with PRU cyst, which shows 62.1% reduction in brain cyst burden in comparison to control group. These results suggest that the combination of self-amplifying RNA and LNP ion would be beneficial to the development of a safe and long-acting vaccine against toxoplasmosis.

Protective immune response in mice induced by a suicidal DNA vaccine encoding NTPase-II gene of Toxoplasma gondii.

DNA-based alphaviral RNA replicon vectors, also called suicidal DNA vectors, have been employed to alleviate biosafety concerns attribution to its ability to induce apoptotic cell death of the transfected cells. Toxoplasma gondii nucleoside triphosphate hydrolase-II (TgNTPase-II), which facilitates the parasite to salvage purines from the host cell for survival and replication, have been demonstrated to be a potential vaccine candidate for toxoplasmosis. Herein, we evaluated the immunogenic potential of a suicidal DNA vaccine encoding TgNTPase-II gene, pDREP-TgNTPase-II, delivered intramuscularly in combination with electroporation. Immunization of mice with pDREP-TgNTPase-II elicited specific humoral responses, with high IgG antibody titers and a mixed IgG1/IgG2a response. The cellular immune response was associated with high level production of IFN-γ, IL-2, IL-10 cytokines and low level IL-4 production as well as the increase of the percentage of CD8+ T cells, indicating that a Th1 predominant response was elicited. Furthermore, mice vaccinated with this suicidal DNA vaccine displayed partial protection against acute infection with the virulent RH strain as well as chronic infection with PRU cyst, which shows 77.7% and 71.4% reduction in brain cyst burden in comparison to PBS and pDREP-eGFP control group, respectively. Based on the cellular and antibody responses, the suicidal DNA vaccine elicited a Th1-predominant immune response against T. gondii challenge.

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED.

Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.

[Effect of rmu-IFN-gamma on the splenic T cell subsets and NK cells of pregnant mice infected with Toxoplasma gondii].

OBJECTIVE: To explore the effect of rmu-IFN-gamma on the change of T cell subsets and natural killer cells of pregnant mice infected with T. gondii. METHODS: Early pregnant mice infected with T. gondii were administered with different doses (1 U/g or 10 U/g) of rmu-IFN-gamma for three days before euthanasia. The numbers of splenic CD4+ and CD8+ T cells and natural killer cells were detected by flow cytometry. RESULTS: Compared with the infected mice that were not treated, the level of splenic CD4+ T cells in mice administered with two doses of rmu-IFN-gamma increased on the day 10, 12, 14 of gestation, while the level of CD8+ T cells decreased on the day 10, 14 of gestation. The ratio of CD4+/CD8+ T cells increased significantly on the day 10, 12, 14 of gestation. Survival days of the two administered groups were longer than those of the infected group. CONCLUSION: A proper dose of rmu-IFN-gamma can reverse the decline of the ratio of T cell subsets, improve the proliferation of NK cells, and so increase the level of peripheral cellular immunity of pregnant mice.

Optimization of a NH4PF6-enhanced, non-organic solvent, dual microextraction method for determination of phthalate metabolites in urine by high performance liquid chromatography.

In conventional ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) procedures, most of the IL disperser remains in the aqueous phase resulting in low recovery for moderately and weakly polar analytes due to the "carry-over effect". Herein, we successfully developed a "NH4PF6-enhanced, non-organic solvent, dual microextraction" method (ANSDM) for pretreatment of phthalate (PAE) metabolites with weak to moderate polarity. This method utilized in situ reaction of NH4PF6 as an ion-exchange reagent and disperser to realize two microextractions after using [C8MIM]PF6 as an extraction solvent and [C4MIM]BF4 as a disperser for conventional DLLME. Single-factor experiments, a two-level full factorial experimental design and central composite design were applied for optimizing operational parameters using 3D response surfaces and contour lines. Under optimized conditions, the newly developed method provided high extraction recoveries (93.8-99.1%) and low LODs (ca. 0.3µgL(-1)) for three phthalate metabolites in human urine. The primary advantages of the ANSDM method include: (1) integration of in situ reaction and conventional DLLME techniques to effectively extract both weak and moderately polar pollutants simultaneously; (2) non-organic solvent use in the microextraction procedure making the process safer and more environmental friendly; and (3) a time-saving, simple operation that is fully compatibility with HPLC analysis. To the best of our knowledge, our group is the first to develop the "non-organic solvent, dual microextraction" method and it has great potential as a sample pre-treatment technique for organic pollutants with weak to moderate polarity in biological and environmental matrices.

Comparison of P1 and 16S rRNA genes for detection of Mycoplasma pneumoniae by nested PCR in adults in Zhejiang, China.

INTRODUCTION: Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen that causes respiratory infections in humans. Laboratory tests are important in the diagnosis of M. pneumoniae because of the atypical features in clinical signs and symptoms. Nowadays, both the P1 adhesin gene and 16S ribosomal (r) RNA (rRNA) gene of M. pneumoniae have been widely detected by polymerase chain reaction (PCR). The purpose of the present study was to evaluate the most suitable target in the detection of M. pneumonia via nested PCR. METHODOLOGY: Both the P1 adhesin gene and 16S rRNA gene for nested PCR reaction conditions were optimized through an orthogonal test and single-factor experiment. Then, the sensitivity of the two sets of targets was evaluated. Finally, based on the optimal conditions, 55 clinical samples of throat swabs collected from adult patients in 2013 were examined by established nested PCR. RESULTS: The results revealed that PCR detection of the 16S rRNA gene was more sensitive than the P1 adhesin gene because the detection limits for both the P1 gene and 16S rRNA gene were at least 100 fg and 10 fg of M. pneumoniae DNA, respectively. Furthermore, the positive rate for detection of the 16S rRNA gene (30/55; 54.5%) was higher than that of the P1 adhesin gene (25/55; 45.5%). CONCLUSION: Our results indicate that the 16S rRNA gene is more suitable for diagnosis of M. pneumoniae infection than the P1 adhesin gene due to its higher sensitivity and positive rate in clinical samples.

In vitro study of the effects of Angiostrongylus cantonensis larvae extracts on apoptosis and dysfunction in the blood-brain barrier (BBB).

It has been hypothesized that blood-brain barrier (BBB) dysfunction in Angiostrongylus cantonensis infection might be due to the apoptosis of the hosts' BBB cells. Here, we evaluated this hypothesis through several methods, all based on an in vitro mouse BBB model consisting of primary culture brain microvascular endothelial cells (BMECs) and brain astrocytic cells (BACs). In the present study, a four-hour percolation and HRP permeability experiment showed that A. cantonensis larvae extracts can increase the permeability of the BBB. Apoptosis among BMECs and BACs after exposure to larvae extracts was monitored by TUNEL and annexin-V-FITC/PI double staining. A. cantonensis larvae extracts were found to induce apoptosis in both BMECs and BACs. For this reason, we concluded that the induction of apoptosis might participate in the BBB dysfunction observed during angiostrongyliasis. Improved fundamental understanding of how A. cantonensis induces apoptosis may lead to new approaches to the treatment or prevention of this parasitic disease.

Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II.

The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.